Graphene Layers as Functional Biomaterials. From Fabrication and Characterization to Biological Applications

ABSTRACT The present study explores the influence of graphene layer number on the adhesion, proliferation, and differentiation of L‐929 fibroblast cells. Double‐ and multi‐layer graphene was fabricated using CVD on Cu and then transferred to other types of substrates by the electrochemical delamination method. Scanning electron microscopy and Raman spectroscopy confirmed the formation of a uniform and continuous graphene film. Four‐point probe measurement of double‐layer graphene revealed a higher sheet resistance. SEM and fluorescence microscopy results revealed a higher density of L‐929 cells on multi‐layer graphene, suggesting that multilayer graphene supported a significantly greater number of cells compared with double‐layer graphene. Immunofluorescence assays, focusing on vinculin and α‐SMA detection, reveal a robust cellular adhesion and extensive proliferation on the multi‐layer substrate and suggest that this has the ability to stimulate the transformation of fibroblasts into myofibroblasts, highlighting its beneficial impact on cellular function. This analysis suggests an innovative approach that demonstrates that layer‐number‐dependent structural features of pristine graphene directly modulate cell adhesion and cytoskeletal behavior, highlighting multilayer graphene as a more effective biointerface for early fibroblast activation.